U.S. patent application number 10/352901 was filed with the patent office on 2004-01-15 for method and system for allergy analysis.
This patent application is currently assigned to Hitachi, Ltd.. Invention is credited to Kato, Hirokazu, KIkkawa, Emiko, Narahara, Masatoshi, Saito, Toshiro, Tomita, Hiroyuki.
Application Number | 20040009499 10/352901 |
Document ID | / |
Family ID | 30112740 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009499 |
Kind Code |
A1 |
Tomita, Hiroyuki ; et
al. |
January 15, 2004 |
Method and system for allergy analysis
Abstract
Provided are a method and a system for allergy analysis by using
RNA derived from leukocytes extracted from peripheral blood as
such, correcting the observed value, and comparing the balance of
helper T (Th) cells (Th1/Th2) with the number of Th1 cells/the
number of Th2 cells in a nucleic acid sample or the Th1/Th2 data of
a patient and of a normal volunteer. Also provided are a method and
a system for allergy analysis using a highly reproducible and
reliable array having a minimal number of DNA fragments
(oligonucleotides) thereon, which is realized by identifying
essential gene groups for the Th1/Th2 assay. According to the
present invention, it is possible to analyze the existence of a
specific cell in a sample containing several types of cells without
isolating the cell of interest.
Inventors: |
Tomita, Hiroyuki;
(Tachikawa, JP) ; Saito, Toshiro; (Hatoyama,
JP) ; Narahara, Masatoshi; (Sayama, JP) ;
Kato, Hirokazu; (Hatoyama, JP) ; KIkkawa, Emiko;
(Kokubunji, JP) |
Correspondence
Address: |
Stanley P. Fisher
Reed Smith LLP
Suite 1400
3110 Fairview Park Drive
Falls Church
VA
22042-4503
US
|
Assignee: |
Hitachi, Ltd.
|
Family ID: |
30112740 |
Appl. No.: |
10/352901 |
Filed: |
January 29, 2003 |
Current U.S.
Class: |
435/6.11 ;
702/20 |
Current CPC
Class: |
G16B 25/10 20190201;
G16B 25/00 20190201; C12Q 1/6883 20130101; C12Q 2600/158
20130101 |
Class at
Publication: |
435/6 ;
702/20 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 2002 |
JP |
2002--204785 |
Claims
What is claimed is:
1. A method for allergy analysis based on the balance of Th1 cells
to Th2 cells ratio (Th1/Th2), comprising the steps of: labeling a
nucleic acid sample derived from human peripheral blood with
fluorescence; performing hybridization of the nucleic acid sample
labeled with fluorescence with a plurality of probes, wherein the
plurality of probes correlate with genes which contain genes that
are expressed specifically in Th cells, genes that are expressed
specifically in Th1 cells, or genes that are expressed specifically
in Th2 cells, or fragments thereof; detecting the presence or
absence of hybridization by the fluorescence as the expression
level; assaying the expression levels of one or more genes that are
expressed specifically in Th1 cells and in Th2 cells respectively
based on detected fluorescence intensities; and comparing (the
number of Th1 cells)/(the number of Th2 cells) in the nucleic acid
sample or the Th1/Th2 data of a patient and of a normal volunteer
with (the expression levels of one or more genes that are expressed
specifically in Th1 cells)/(the expression levels of one or more
genes that are expressed specifically in Th2 cells) as Th1/Th2.
2. The method for allergy analysis according to claim 1, wherein
when the gene which correlate with the plurality of probes further
contain genes that are expressed specifically in cells other than
Th cells and the nucleic acid sample is also expressed in cells
other than Th cells, as the expression level for one or more genes
that are expressed specifically in Th1 cells and that in Th2 cells,
the value of (fluorescence intensity).times.(.alpha./(.alpha.+1))
(.alpha.: the expression level for one or more genes that are
expressed specifically in Th cells/the expression level for one or
more genes that are expressed specifically a cell other than Th
cells) is employed, respectively.
3. The method for allergy analysis according to claim 2, wherein as
the expression level for one or more genes that are expressed
specifically in Th1 cells and that in Th2 cells, a sum or an
average of fluorescence intensities is employed, respectively, and
the product with .beta., a normalizing function to realize
Th1/Th2=1 when Th1:Th2 is 1:1, is used as the value of Th1/Th2.
4. The method for allergy analysis according to claim 2, wherein as
the expression level for one or more genes that are expressed
specifically in Th1 cells and that in Th2 cells, a median of the
fluorescence intensity ranking among the plurality of probes is
employed, respectively, and the product with .beta. is used as the
value of Th1/Th2.
5. The method for allergy analysis according to claim 2, wherein
the cell other than Th cells is at least one type of cells selected
from neutrophils, eosinophils, basophils, monocytes, macrophages, B
cells, and NK cells.
6. A system for allergy analysis based on the balance of Th1 cells
to Th2 cells ratio (Th1/Th2) comprising: an oligonucleotide array
having a plurality of probes immobilized thereon, wherein the
plurality of probes correlate with genes comprising genes that are
expressed specifically in Th cells, genes that are expressed
specifically in Th1 cells, and genes that are expressed
specifically in Th2 cells; a detection means for detecting, as the
expression level, the fluorescence of a nucleic acid sample that is
hybridized with the probes; a data storage means for storing data
on the number of Th1 cells and the number of Th2 cells in the
nucleic acid sample or Th1/Th2 for a patient and for a normal
volunteer; a data storage means for storing data on the expression
level for one or more genes that are expressed specifically in Th1
cells and in Th2 cells based on detected fluorescence intensities;
and a computer for comparing (the number of Th1 cells)/(the number
of Th2 cells) in the nucleic acid sample or the Th1/Th2 data of a
patient and of a normal volunteer with the (expression level for
one or more genes that are expressed in Th1 cells)/(the expression
level for one or more genes that are expressed in Th2 cells) as
Th1/Th2.
7. The system for allergy analysis according to claim 6, wherein
when the gene which correlate with the plurality of probes further
contain genes that are expressed specifically in cells other than
Th cells and the nucleic acid sample is also expressed in cells
other than Th cells, as the expression level for one or more genes
that are expressed specifically in Th1 cells and that in Th2 cells,
the value of (fluorescence intensity).times.(.alpha./(.alpha.+1))
(.alpha.: the expression level for one or more genes that are
expressed specifically in Th cells/the expression level for one or
more genes that are expressed specifically in cells other than Th
cells) is employed, respectively.
8. The system for allergy analysis according to claim 7, wherein as
the expression level for one or more genes that are expressed
specifically in Th1 cells and that in Th2 cells, a sum or an
average of the fluorescence intensities is employed, respectively,
and the product with .beta., a normalizing function to realize
Th1/Th2=1 when Th1:Th2 is 1:1, is used as the value of Th1/Th2.
9. The system for allergy analysis according to claim 7, wherein as
the expression level for one or more genes that are expressed
specifically in Th1 cells and that in Th2 cells, a median of the
fluorescence intensity ranking among the plurality of probes is
employed, respectively, and the product with .beta. is used as the
value of Th1/Th2.
10. The system for allergy analysis according to claim 7, wherein
the cell other than Th cells is at least one type of cells selected
from neutrophils, eosinophils, basophils, monocytes, macrophages, B
cells, and NK cells.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method and a system, for
analyzing the existence of a specific cell in a sample containing
several types of cells through gene expression by using an
oligonucleotide array. More particularly, the present invention
relates to a method and a system for allergy analysis for simple
evaluation of allergy levels using an oligonucleotide array.
BACKGROUND ART
[0002] Recently, increases in patients suffered from life-style
related diseases or atopic or allergic diseases have become one
factor for increased medical expenditures for people. Up to the
present, administration of topical steroidal drugs has been the
therapeutic method for allergic diseases, although this cannot
inhibit the symptoms completely. On the other hand, desensitizing
therapy has recently drawn attention. This method is carried out by
administering an antigen extract (e.g., mite antigen) sublingually
or the like. Desensitizing therapy is a therapeutic method for
attenuating allergic reactions by repeatedly administering antigens
to patients with specific allergens, in which mite, cedar pollen,
or the like is used as the antigen. Compared to other types of drug
therapy, side effects caused by desensitizing therapy is smaller,
and if it works successfully, significant therapeutic effects can
be attained. Thus, it is very beneficial for allergy patients.
However, the success or failure of the therapy depends on the
optimization of the antigen liquid concentration. The higher the
antigen liquid concentration, the better the therapeutic effect.
Side effects, however, should not be induced. At present, there is
no method for determining the optimal antigen liquid concentration
which varies for each individual. Further, there is no diagnostic
index for determining whether the therapy should be continued or
switched to other therapy when therapeutic effects are not
apparent.
[0003] Allergy, however, is a serious symptom with which many
patients are afflicted. It would be beneficial if allergy can be
easily treated or diagnosed by general practitioners and the like,
not to mention specialized medical institutions. From such a point
of view, the development of diagnostic instruments, which are
targeted to assess for appropriate levels of therapeutic measures
for allergies and their therapeutic effects has been awaited.
[0004] It is considered that the benefits of the desensitizing
therapy mentioned above are affected because the balance of helper
T cells is changed and the migration of inflammatory cytokines is
inhibited. Helper T cells can be classified into cell populations
referred to as Th1 and Th2 depending on the types of cytokines
generated therefrom, and they regulate different immune responses.
Th1 cells are mainly involved with phylaxis through the
cell-mediated immune responses against intracellular parasitic
microorganisms, and Th2 cells are mainly involved with phylaxis
through the humoral immune responses against extracellular
parasitic microorganisms. These two types of cell populations
maintain the equilibrium with each other through the cytokines
produced by the other, thereby regulating each other to prevent
excessive reactions from taking place. Many diseases such as
allergic diseases developing due to the action of the Th1 or Th2
are considered to develop from imbalance in Th cells.
[0005] Up to the present, genes that are expressed differently in
Th1 and in Th2 have been determined by DNA chips or Serial Analysis
of Gene Expression (SAGE), and reported. Rogge et al. reported 215
genes that are expressed differently in Th1 and in Th2 using DNA
chips having 6,000 genes placed thereon (Rogge, L. et al.,
Transcript imaging of the development of human T helper cells using
oligonucleotide arrays, Nature Genetics 25, 96-101, 2000). Also,
Nagai et al. (Nagai, S et al., Comprehensive gene expression
profile of human activated Th1- and Th2-polarized cells,
International Immunology 13, 367-376, 2001) analyzed 20,000 or more
transcripts in Th1 cells and in Th2 cells respectively by SAGE, and
reported 66 genes with higher levels of expression (critical rate
of 5% or lower) in Th1 cells and 14 genes with higher levels of
expression (critical rate of 5% or lower) in Th2 cells. Thus, from
both reports, it is recognized that a wide variety of genes, which
encode proteins such as cytokine, growth factor, receptor,
transcription factor, cell adhesion-associated protein, cell
migration-associated protein, ion channel, transporter,
apoptosis-associated protein, signal transduction-associated
protein, or metabolic pathway-associated protein, are expressed
differently in Th1 cells and in Th2 cells. It should be noted that
except for a few, the genes mentioned in these two reports are
substantially different from each other.
[0006] In principle, a Th1/Th2 balance can be assayed by observing
the distribution of the expression levels of the genes reported
above. Such analyses, however, have never been carried out in the
field of medical care. For one reason, the above-mentioned reports
utilize the Th1 and Th2 cells previously isolated from blood by a
given method. In actual medical practice, the amount of blood that
can be obtained from a subject in one sampling is 5 to 10 cc. The
amount of total RNA that can be extracted from this 5 to 10 cc of
blood is not more than 10 .mu.g with some inter-individual
variations. In humans, since not more than 5% of the amount of
total RNA is messenger RNA, the messenger RNA level that can be
extracted from 5 to 10 cc of blood is only 0.5 .mu.g. In addition,
blood contains lymphocytes such as B cells and NK cells,
neutrophils, eosinophils, basophils, and granulocytes such as
monocytes/macrophages, as well as T cells. Accordingly, the
messenger RNA level derived from Th1, Th2 cells that can be
extracted from 5 to 10 cc of blood is 0.05 .mu.g or lower, which is
significantly smaller than the messenger RNA level commonly used in
DNA chips, i.e., 26.0 .mu.g. Specifically, it is technically very
difficult to conduct routine examinations by extracting only Th1
and Th2 cells from the subject and further extracting RNA
therefrom. Also, isolation of Th1 and Th2 cells from blood requires
many processes, which in turn increases a cost in one diagnosis.
Accordingly, it is not preferable from the viewpoint of medical
economics. The recovery in each process cannot be always 100%, and
thus, the amount of RNA that can be used in diagnosis decreases as
the number of pretreatment processes increases.
[0007] In order to overcome the difficulties mentioned above, the
Th1 cells:Th2 cells ratio is preferably determined by using RNA
obtained from whole blood instead of RNA obtained from helper T
cells only.
SUMMARY OF THE INVENTION
[0008] An object of the present invention is to provide a method
for analyzing the existence of a specific cell in a sample
containing several types of cells without isolating the cell of
interest. In one preferred embodiment, the present invention
provides a method and a system for allergy analysis using an
oligonucleotide array with the propriety of therapeutic measures
and the level of their therapeutic effects can be assessed in a
simple manner at low cost with high reliability. In a conventional
analytical method by isolating T cells, T cell isolation and, in
some cases, culture should be conducted. This increases the cost
and time for analysis. Further the distribution of cellular gene
expression may be affected by processes such as T cell isolation
and culture. Thus, it is preferable not to carry out T cell
isolation insofar as conditions permit. Accordingly, the present
invention provides a method and a system for allergy analysis by
using RNA derived from leukocytes extracted from peripheral blood
as such, correcting, assessing the balance of the helper T (Th)
cells (the Th1 cells:Th2 cells ratio (Th1/Th )), and comparing the
value with the number of Th1 cells/the number of Th2 cells in a
nucleic acid sample or Th1/Th2 data of a patient and of a normal
volunteer. It is another object of the present invention to provide
a method and a system for allergy analysis using a highly
reproducible and reliable array having a minimal number of DNA
fragments (oligonucleotides) thereon, which is realized by
identifying gene groups that are essential for the Th1/Th2
assay.
[0009] We have conducted concentrated studies in order to attain
the object of evaluating allergy levels using an oligonucleotide
array. As a result, we found that, when assaying Th1 cells: Th2
cells ratio, the expression of genes in leukocyte: for example, (1)
genes that are expressed specifically in Th cells; (2) genes that
are expressed specifically in cells other than T cells, such as B
cells and monocytes; (3) genes that are expressed equally in Th
cells and in other cells; and (4) genes that are expressed
differently in Th1 cells and in Th2 cells should be assayed
preferably on the same array. In particular, when RNA obtained from
whole blood is used, a preferred method is a method for determining
the Th1 cells:Th2 cells ratio based on the genes which satisfy
conditions (1) and (4) above, i.e., genes that are expressed
specifically in Th cells and expressed differently in Th1 cells and
in Th2 cells, and comprising correction of the expression level of
(4), i.e., the expression level of genes that are expressed
differently in Th1 cells and in Th2 cells using the expression
levels of genes of (1) and (2), i.e., the expression level of genes
that are expressed specifically in Th cells and genes that are not
expressed in T cells and the expression level of genes of (3) that
are expressed equally in Th cells and in other cells.
[0010] More specifically, the present invention provides a method
for allergy analysis by observing the balance of the Th1 cells:Th2
cells ratio (Th1/Th2), comprising the steps of:
[0011] labeling a nucleic acid sample derived from human peripheral
blood with fluorescence;
[0012] performing hybridization of the nucleic acid sample labelled
with fluorescence with a plurality of probes, which contain genes
that are expressed specifically in Th cells, genes that are
expressed specifically in Th1 cells, and genes that are expressed
specifically in Th2 cells;
[0013] detecting the presence or absence of hybridization by the
fluorescence as the expression level;
[0014] assaying expression levels of one or more genes that are
expressed specifically in Th1 cells and in Th2 cells respectively
based on detected fluorescence intensity; and
[0015] comparing (the number of Th1 cells)/(the number of Th2
cells) in the nucleic acid sample or the Th1/Th2 data of a patient
and of a normal volunteer with (Th1/Th2)=(the expression levels of
one or more genes that are expressed specifically in Th1
cells)/(the expression levels of one or more genes that are
expressed specifically in Th2 cells), thereby analyzing
allergy.
[0016] When complementary DNA (cDNA) is synthesized by reverse
transcription using, as a template, RNA obtained from leukocytes or
T cells derived from human peripheral blood, cDNA is labelled by
incorporating a labelling substance at the time of binding or
extension, and the labelled cDNA can be used as a nucleic acid
sample.
[0017] In this specification, "genes that are expressed
specifically in cell A" refers to genes, that have expression
levels that are statistically significant in cell A but below the
lower limit of the measurable range or are statistically
insignificant in cells other than cell A, which are contained in
the specimen, according to the observation using, for example, a
DNA chip or assay methods such as RT-PCR.
[0018] In the present invention, the number of genes that are
expressed specifically in Th1 and in Th2, the number of genes that
are expressed specifically in cells other than Th cells, and the
number of genes that are expressed differently in Th1 and in Th2,
are respectively 1 or more and preferably in the range of 50 to
200, for accurate analysis. The use of a larger number of genes
enables more accurate analysis. If the number is too large,
however, workloads in experiments or correction increase. Thus, the
number of genes in the above-stated range is sufficient to obtain
reliable values. Cells other than T cells include at least one cell
selected from neutrophil, eosinophil, basophil, monocyte,
macrophage, B cell, and NK cell.
[0019] In this specification, the term "expression level" refers to
cellular levels of messenger RNA or cellular levels of protein. A
sum, average, or median of the expression levels of all genes is
used as the expression level of a plurality of genes. When the
expression is assayed based on the fluorescence intensity, the
median of the fluorescence intensity ranking among the plurality of
probes can be employed.
[0020] According to the method of the present invention, when genes
corresponding to s plurality of probes also contain genes that are
expressed specifically in cells other than Th cells and the nucleic
acid sample is also expressed in cells other than Th cells, the
expression levels of one or more genes that are expressed
specifically in Th1 cells and in Th2 cells can be respectively
(fluorescence intensity).times.(.alpha./(.alpha.+1)) (.alpha.: the
expression levels of one or more genes that are expressed
specifically in Th cells/the expression levels of one or more genes
that are expressed specifically in cells other than Th cells).
[0021] Further, the expression level for one or more genes that are
expressed specifically in Th1 cells and in Th2 cells can be a sum
or an average of the fluorescence intensity values of respective
genes and the product with .beta. (a normalizing function to
realize Th1/Th2=1 when Th1:Th2 is 1:1) can be Th1/Th2.
[0022] Alternatively, the expression level for one or more genes
that are expressed specifically in Th1 cells and in Th2 cells can
be a median of the fluorescence intensity ranking among the
fluorescence intensity values of the plurality of probes,
respectively, and the product with .beta. can be Th1/Th2.
[0023] The method according to the present invention can be used
for assessing a disease status and/or disease level by, for
example, assaying Th1/Th2 in the sample. The "specific state"
refers to a control condition such as a normal state when, for
example, a disease state and/or level of allergy and the like is to
be analyzed. If Th1/Th2 is 1 in a normal state, statistical
significance based on deviations from 1 is evaluated. Examples of
statistical techniques that are used in the present invention
include Bayesian estimation and Neyman-Pearson's estimation,
without particular limitations.
[0024] In this specification, the term "expressed equally" refers
to the level of gene expression with .+-.10%. Specifically, when
the difference in the expression levels of a certain gene in two or
more cells is within .+-.10%, the gene is referred to be "expressed
equally" in the two or more cells.
[0025] In the method of the present invention, the number of genes
used that are expressed equally in all of a pluraliy types of cells
is 1 or more, and preferably 10 to 50.
[0026] According to the method of the present invention, changes
with time in the ratio of the number of one or several types of
cells to the number of entire cells can be analyzed, and whether or
not the number of a specific type of cell increased or decreased in
a sample comprising a plurality of types of cells can be analyzed
without isolating the cell of interest.
[0027] Analysis of the Th1/Th2 value in a sample by the above
method without the separation of cells from one another enables
rapid assessment of whether, with respect to normal values, Th is
imbalanced or not or the degree of imbalance thereof. Thus,
information useful for therapy can be provided. Further, previous
data accumulation for a patient and for a normal volunteer can
express the correlation between the Th1/Th2 value and the disease
rate as a threshold model. Therapeutic measures for whether or not
the subject should be treated can be also determined based on the
Th1/Th2 value obtained in each examination. Furthermore, the
diagnostic accuracy can be enhanced by using an assessment method
based on Bayesian Statistics or determination.
[0028] The present invention further provides a system for allergy
analysis by observing the balance of the Th1 cells:Th2 cells ratio
(Th1/Th2) comprising:
[0029] an oligonucleotide array having a plurality of probes, which
correlate a plurality of genes comprising genes that are expressed
specifically in Th cells, genes that are expressed specifically in
Th1 cells, and genes that are expressed specifically in Th2 cells,
immobilized thereon;
[0030] a detection means for detecting, as the expression level,
the fluorescence of a nucleic acid sample hybridized with the
probes;
[0031] a data storage means for storing data on the number of Th1
cells and the number of Th2 cells in the nucleic acid sample or
Th1/Th2 of a patient and of a normal volunteer;
[0032] a data storage means for storing data on the expression
levels of one or more genes that are expressed specifically in Th1
cells and in Th2 cells based on the detected fluorescence
intensities; and
[0033] a computer for comparing (the number of Th1 cells)/(the
number of Th2 cells) in the nucleic acid sample or the Th1/Th2 data
of a patient and of a normal volunteer with (Th1/Th2)=(the
expression level for one or more genes that are expressed in Th1
cells)/(the expression level for one or more genes that are
expressed in Th2 cells).
[0034] This specification includes part or all of the contents as
disclosed in the specification and/or drawings of Japanese Patent
Application No. 2002-204785, which is a priority document of the
present application.
BRIEF DESCRIPTION OF DRAWINGS
[0035] FIG. 1 shows an example of positioning probe DNA on a
substrate.
[0036] FIG. 2 shows a general structure of a DNA chip.
[0037] FIG. 3 shows an example in which a sample containing only
Th1 and Th2 cells is assayed to evaluate Th1/Th2.
[0038] FIG. 4 shows an example in which a sample containing
monocytes in addition to Th1 and Th2 cells are assayed to evaluate
Th1/Th2.
[0039] FIG. 5 schematically shows the DNA chip production.
[0040] FIG. 6 shows a flow chart for the DNA chip production.
[0041] FIG. 7 schematically shows the DNA chip data analysis.
[0042] FIG. 8 shows a flow chart for the DNA chip data
analysis.
[0043] FIG. 9 shows a flow chart indicating a method for selecting
a suitable formula from among formulae 4-1 to 4-6.
DESCRIPTION OF REFERENCE NUMERALS
[0044] 1. substrate
[0045] 2. probe DNA immobilized region
[0046] 11. immobilized regions of DNA fragments (probe DNA) having
gene sequences of genes that are expressed differently in Th1 cells
and in Th2 cells or complementary sequence strands thereof
[0047] 12. probe DNA of genes that are expressed specifically in Th
cells
[0048] 13. immobilized regions of probe DNA of genes that are not
expressed very much in Th cells but are expressed specifically in
cells other than T cells, such as B cells and monocytes
[0049] 14. immobilized region of probe DNA of genes that are
expressed equally in Th cells and in other cells
[0050] 111. immobilized region of probe DNA of genes that are
expressed differently in Th1 cells and in Th2 cells and the
expression level in Th1 cells is higher than that in Th2 cells
[0051] 112. immobilized region of probe DNA of genes that are
expressed differently in Th1 cells and in Th2 cells and the
expression level in Th2 cells is higher than that in Th1 cells
[0052] 21. fluorescence detector
[0053] 22. DNA probe
[0054] 23. fluorescence-labelled gene
[0055] 24. support
[0056] 25. public database
[0057] 26. in-house database
[0058] 27. computer for designing probe sequence
[0059] 28. probe sequence
[0060] 29. nucleic acid synthesizer
[0061] 30. oligonucleotide probe set
[0062] 31. spotter
[0063] 32. computer for controlling spotter
[0064] 33. chip (production in progress)
[0065] 34. chip (produced)
[0066] 35. fluorescence-labelled sample
[0067] 36. chip
[0068] 37. fluorescence detector
[0069] 38. computer for controlling fluorescence detector
[0070] 39. experimental data
[0071] 40. computer for analyzing experimental data
PREFERRED EMBODIMENTS OF THE INVENTION
[0072] The present invention is hereafter described in more
detail.
[0073] In this specification, an "internal control gene for
proofreading" refers to, for example, a house keeping gene. The
house keeping gene encodes, for example, a structural protein that
is necessary for cellular survival or an enzyme in the energy
metabolic system, and it is considered to be expressible in any
cell with various types of differentiation. Examples thereof
include .beta.-actin, GAPDH, HPRT, alpha-tublin, a transferrin
receptor, and ubiquitin. Since these are already contained in the
sample from the subject, such as leukocytes, they can be internal
controls at the time of proofreading. These internal controls are
already contained in the sample and become controls at the time of
proofreading.
[0074] In this specification, an "external control gene for
proofreading" refers to, for example, when a human is to be
assayed, a gene sequence from plants, microorganisms, insects, or
the like that is absent in humans. Examples thereof include
Arabidopsis thaliana gene, plasmid DNA, bacteriophage DNA, and
Firefly luciferase gene. Since it is not contained in the sample
from the subject, such as leukocytes, it can be an external control
at the time of proofreading when a known concentration of this
external control gene is externally added at the time of assay. The
external control is not previously present in the sample, and thus,
it becomes a control at the time of proofreading by being
externally and separately added.
[0075] T cells are described. T cells are also referred to as
T-lymphocytes, they are a subpopulation that accounts for 60 to 80%
of the peripheral blood lymphocytes, and they are derived from bone
marrow stem cells. Most thereof mature in the thymes gland. Many
mature T cells become helper T (Th) cells or killer T cells. The
helper T cells produce various cytokines by antigen stimulation,
and act on B cells, T cells, macrophages, and the like to induce
acceleration and potentiation of immune response. The killer T
cells are capable of recognizing and attacking transplants, virus
infected cells, and tumor cells. Scientifically, it is known that
the STAT4, IL12RB2, TBX21, IFNG, ERM, IL18, JAK2, TYK2, IL18R, IL2,
HLX, and IRF1 genes are essential for Th1 cell differentiation.
SCYC1, SPP1, MIP1A, MIP1B, PRF1, CTNNB1, CXCR3, CCR5, RANTES, and
NFKB are genes that are not always necessary in Th1 cell
differentiation but scientifically expected to be involved
therewith, and to be expressed differently in Th1 cells and in Th2
cells. Also known is the SOCS1 gene that is considered to inhibit
differentiation to Th2 cell upon Th1 cell differentiation.
[0076] Similarly, it is scientifically known that the STAT6, GATA3,
MAF, IL13IL5, IL4, IL6, IL10, NFAT, and NIP45 genes are essential
in Th2 cell differentiation. FUS, ILF2, E3-EPF, PSG, CRTH2, and
CCR4 are genes that are not always necessary in Th2 cell
differentiation but scientifically expected to be involved
therewith, and to be expressed differently in Th1 cells and in Th2
cells. Also known is the SOCS3 gene that is considered to inhibit
differentiation to Th1 cell upon Th2 cell differentiation.
[0077] It is scientifically considered that the IL12RB1, IL4RA,
IL3, and GM-CSF genes are expressed substantially equally in Th1
cells and in Th2 cells.
[0078] The balance maintained by the cytokines generated by Th1
cells and Th2 cells can be expressed at a genetic level as follows.
If several groups of Th1/Th2 associated genes are switched, the
level of the Th1/Th2 associated protein are either increased or
decreased, and the balance of these protein activities, as a whole,
is considered to regulate biofunctions. Specifically, abnormal
switching of groups of Th1/Th2 associated genes results in
imbalance of protein activities. This manifests as a regulatory
abnormality of biofunctions such as Th1/Th2. Switching of genes is
regulated by, for example, an increase or decrease in the gene
expression level. The gene expression level can be assayed by
employing a messenger RNA level or a protein level as an index. In
currently available techniques, the assay can be carried out much
more easily by employing, as an index, the messenger RNA level
instead of the protein level. For simple evaluation of allergy, an
increase/decrease in the expressed messenger RNA level of a
plurality of Th1/Th2 associated genes may be simultaneously
observed. To this end, the DNA array (this may be referred to as an
"oligonucleotide array") is most suitable when the number of
associated genes is more than 100 while the DNA array or the
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is
suitable when the number of associated genes is not more than 100.
In the present invention, the "DNA array" and the "oligonucleotide
array" include those prepared by synthesizing oligonucleotides on a
support and those prepared by spotting amplified gene fragments
(oligonucleotides) on a support. It should be noted that the
processes for producing those are not specified by their names.
[0079] The DNA array (oligonucleotide array) comprises a plurality
of DNA fragments (oligonucleotides) immobilized on a substrate.
Each oligonucleotide independently corresponds to a different gene.
When assaying, messenger RNA derived from an assay sample is
employed as a template for reverse transcription, thereby
synthesizing a complementary DNA (cDNA) fragment. When only a small
amount of sample is available, it may be amplified by PCR or the
like. At the time of the reverse transcription or PCR, a suitable
labelling substance such as a fluorescent substance is incorporated
upon binding or extension, thereby labelling the cDNA fragment
(hereinafter, cDNA with a label is referred to as the "labelled
cDNA").
[0080] The oligonucleotide that is immobilized on a substrate is
bound complementarily with the labelled cDNA fragment. Each
oligonucleotide is immobilized on the substrate at different
coordinates. If the types of oligonucleotides immobilized at given
coordinates can be previously identified, a decrease/increase in
messenger RNA for a plurality of individual genes can be
simultaneously assayed.
[0081] RT-PCR is a method for assaying the expression of the
subject genes by employing genes with small intercellular
variations in the expression level (e.g., house keeping genes) as a
standard. Since the messenger RNA derived from an assay sample can
be amplified by RT-PCR, a smaller amount of sample than the DNA
array method suffices for the assay.
[0082] FIG. 5 shows a general scheme for the production of DNA
chips, and the flow chart therefor is shown in FIG. 6. Gene
information (such as sequence information and functional
information) are first obtained from public database 25 accessible
from a network such as the Internet or in-house database 26. Based
on these pieces of gene information, probe sequences for specific
genes are designed, and these sequences are designed by computer 27
for designing probe sequences. The computed nucleotide sequence 28
is input into nucleic acid synthesizer 29 to synthesize
oligonucleotide probes. The synthesized probe set 30 is spotted on
a support of DNA chip (production in progress) 33 using spotter 31,
thereby preparing DNA chip 34.
[0083] FIG. 7 schematically shows the analysis of DNA chip data,
and the flow chart therefor is shown in FIG. 8. A nucleic acid
sample obtained from the subject is first labelled with a
fluorescent label to prepare fluorescence-labelled sample 35. The
fluorescence-labelled sample 35 is then hybridized onto chip
(oligonucleotide array) 36 comprising a plurality of probes
(oligonucleotides), immobilized thereon, which can be hybridized
with a nucleic acid sample in a sequence-specific manner. After
hybridization, the chip 36 is screened using fluorescence detector
(detection means) 37 to obtain fluorescence levels (fluorescence
intensity) corresponding to each probe. The obtained fluorescence
level is stored in computer 38 for regulating fluorescence detector
(data storing means) as experimental data 39 (data on distribution
of gene expression). Subsequently, the experimental data 39 is
transferred to computer 40 for analyzing experimental data to
perform correction or statistical analysis by the formulae by the
method disclosed in this specification or the like. The results of
the analysis are provided to the analyzer in the form of a screen
output or report output.
[0084] When a nucleic acid sample derived from a subject with
allergy or with suspected allergy is used as a sample, the allergy
status and/or level can be analyzed by the above method or
system.
[0085] The present invention provides a method and a system of
analysis using an oligonucleotide array comprising a plurality of
oligonucleotides with different nucleotide sequences immobilized
onto a support at different locations, wherein the oligonucleotides
have a nucleotide sequence comprising at least 20 consecutive
nucleotides of the genes according to (1) to (4) below or
complementary sequence strands of the genes described below.
[0086] More specifically, groups (1) to (4) refer to:
[0087] (1) an oligonucleotide derived from genes that are expressed
specifically in cell 1 and in cell 2 or an oligonucleotide that is
complementary to the oligonucleotide mentioned just above;
[0088] (2) an oligonucleotide derived from genes that are expressed
specifically in cell k (k=3, 4, . . . , n) or an oligonucleotide
that is complementary to the oligonucleotide mentioned just
above;
[0089] (3) an oligonucleotide derived from genes that are expressed
equally in all the n types of cells or an oligonucleotide that is
complementary to the oligonucleotide mentioned just above; and
[0090] (4) an oligonucleotide derived from genes that are expressed
differently in cell 1 and in cell 2 or an oligonucleotide that is
complementary to the oligonucleotide mentioned just above.
[0091] The number of oligonucleotides to be immobilized which
belong to each group in the oligonucleotide array is not
particularly limited. Preferably, the numbers thereof for groups
(1), (2), and (3) are respectively in the range of 10 to 50, and in
the range of 50 to 200 for group (4).
[0092] When immobilizing oligonucleotides on a substrate, they are
preferably positioned separately depending on their groups. Thus,
the assay results obtained by the oligonucleotide array can be
instantaneously and visually understood and assessed by the
examiner.
[0093] The present invention also provides to a method and a system
of analysis using an oligonucleotide array comprising
oligonucleotides (equivalent to (4)) immobilized thereon, each
having a nucleotide sequence comprising at least 20 consecutive
nucleotides in genes that are expected to be expressed differently
in Th1 cells and in Th2 cells or a complementary sequence thereof.
Genes that are expressed differently in Th1 cells and in Th2 cells
include a wide variety of genes, which encode cytokine, chemokine,
growth factor, transcription factor, apoptosis-associated protein,
proteolysis-associated protein, signal transduction-associated
protein, enzyme, ion channel, transporter, metabolism-associated
protein, cell adhesion-associated protein, cell
migration-associated protein, and allergic protein. Specifically,
in terms of Symbol designations of the Unigene, these genes
include, for example, ADCY7, AGXT, AKAP1, ALDH3B1, ANXA3, ARCN1,
AREG, ARRB2, ATP1A1, ATP6D, ATP6F, BAK1, BIRC3, CACNB3, CASP8,
CCR4, CCR5, CD2, CD38, CD3D, CD6, CD69, CD97, CDH3, CEBPD, CNK,
COPS3, COX5B, CRI1, CSF2, CST3, CTNNB1, CXCR3, DAF, DNTT, DPP4,
DTR, E2-EPF, E2F4, EBI2, EDG1, EGR2, ERM.ETS1, FACL1, FGD1, FGFR1,
FUS, FLJ14639, FLJ20746, FOS, FOXG1A, FUS, GATA1, GATA3, GBP1,
GBP2, GC20, GCH1, GNLY, GOT1, GPR6, GPR9, GZMB, GZMK, HIF1A, HLX,
HNRPA1, HOXA1, HSP105B, HSPA1A, HSPCA, HSXIAPAF1, HT013, ICAM2,
ICSBP1, IFI35, IFNG, IL2, IL4, IL5, IL6, IL10, IL10RA, IL12RB2,
IL13, +IL18R1, IL2, IL3, IL9, IL13, IL18, IL18R, ILF2, IRF1, IRF7,
ITGB7, ITK, JAK2, JUN, KCNB2, KCNK3, KIAA0239, KLK6, KSR, LDHB,
LEP, LIFR, LOC51042, LOC64116, LRP8, LTA, LTB, MAF, MAP2K2, MAP3K5,
MIG, MIP, MKNK1, MRF-1, MSN, MT1H, MYB, NDUFB5, NFATC1, NFKB, NKG7,
NME4, NR4A2, NT5, OSM, P2RX5, PDCD5, PDE4B, PEF, PEMT, PheHB, IM1,
PLAUR, PP, PPIF, PPP1R2, PPP2R5D, PRDX1, PRF1, PRKCB1, PRKCH,
PSCDBP, PSMB4, PSMC4, PSMC6, PTB, PTGER4, PTPRA, PTPRZ1, RAB32,
RDBP, RORA, RPS24, SCYA15, SCYA17, SCYA3, SCYA4, SCYA5, SCYC1, SDH,
SELL, SERPINB1, SET, SF3A3, SFRS10, SFRS5, SLA, SOCS1, SOCS3, SPP1,
SRM300, STAT1, STAT4, STAT6, STIP1, TAF2S, TANK, TAP1, TBX21,
TBXA2R, TIEG, TIMP1, TNFRSF1A, TNFRSF1B, TNFSF10, TNFSF5, TYK2,
UROD, USF2, USP12, USP7, XBP1, YME1L1, and YR-29. Examples of these
genes are shown in Tables 1 to 6 together with their accession
numbers in the GenBank.
1 TABLE 1 Unigene Symbol RefSec(GenBank) No. ADCY7 NM_001114 AGXT
NM_000030 AKAP1 NM_003488 ALDH3B1 NM_000694 ANXA3 NM_005139 ARCN1
NM_001655 AREG NM_001657 ARRB2 NM_004313 ATP1A1 NM_000701 ATP6D
L05087 ATP6F NM_004047 BAK1 NM_001188 BIRC3 NM_001165 CACNB3
NM_000725 CASP8 NM_001228 CD2 NM_001767 CD38 NM_001775 CD3D
NM_000732 CD6 NM_006725 CD69 NM_001781 CD97 NM_001784 CDH3
NM_001793 CEBPD NM_005195 CNK NM_004073 COPS3 NM_003653 COX5B
NM_001862 CRI1 NM_014335 CSF2 NM_000758 CST3 NM_000099 CTNNB1
NM_001904
[0094]
2 TABLE 2 Unigene Symbol RefSec(GenBank) No. DAF NM_000574 DNTT
NM_004088 DPP4 NM_001935 DTR NM_001945 E2-EPF NM_014501 E2F4
NM_001950 EBI2 NM_004951 EDG1 NM_001400 EGR2 NM_000399 ETS1
NM_005238 FACL1 NM_001995 FGD1 NM_004463 FGFR1 NM_000604 FLJ20746
NM_019025 FOS NM_005252 FOXG1A NM_004471 FUS NM_004960 GATA1
NM_002049 GATA3 NM_002051 GBP1 NM_002053 GBP2 NM_004120 GC20
NM_005875 GCH1 NM_000161 GNLY NM_006433 GOT1 NM_002079 GPR6
NM_005284 GPR9 NM_001504 GZMB NM_004131 GZMK NM_002104 HIF1A
NM_001530
[0095]
3TABLE 3 Unigene Symbol RefSec(GenBank) No. HNRPA1 NM_002136 HOXA1
NM_005522 HSP105B NM_006644 HSPA1A NM_005345 HSPCA NM_005348
HSXIAPAF1 NM_017523 HT013 NM_018474 ICAM2 NM_000873 ICSBP1
NM_002163 IFI35 U72882 IFNG NM_000619 IL10RA NM_001558 IL12RB2
NM_001559 IL13 NM_002188 IL18R1 NM_003855 IL2 NM_000586 IL3
NM_000588 IL9 NM_000590 ILF2 NM_004515 IRF1 NM_002198 IRF7
NM_001572 ITGB7 NM_000889 ITK NM_005546 JUN NM_002228 KCNB2
NM_004770 KCNK3 NM_002246 KIAA0239 NM_015288 KLK6 NM_002774 KSR
U43586 LDHB NM_002300
[0096]
4 TABLE 4 Unigene Symbol RefSec(GenBank) No. LEP NM_000230 LIFR
NM_002310 LOC51042 NM_015871 LOC64116 NM_022154 LRP8 NM_017522 LTA
NM_000595 LTB NM_002341 MAP2K2 NM_030662 MAP3K5 NM_005923 MIG
NM_002416 MKNK1 NM_003684 MRF-1 M62324 MSN NM_002444 MT1H NM_005951
MYB NM_005375 NDUFB5 NM_002492 NFATC1 NM_006162 NKG7 NM_005601 NME4
NM_005009 NR4A2 NM_006186 NT5 NM_002526 OSM NM_020530 P2RX5
NM_002561 PDCD5 NM_004708 PDE4B NM_002600 PEF NM_012392 PEMT
NM_007169 PheHB NM_005687 PIM1 NM_002648 PLAUR NM_002659
[0097]
5 TABLE 5 Unigene Symbol RefSec(GenBank) No. PP NM_021129 PPIF
NM_005729 PPP1R2 NM_006241 PPP2R5D NM_006245 PRDX1 NM_002574 PRF1
NM_005041 PRKCB1 NM_002738 PRKCH NM_006255 PSCDBP NM_004288 PSMB4
NM_002796 PSMC4 NM_006503 PSMC6 NM_002806 PTB NM_002819 PTGER4
NM_000958 PTPRA NM_002836 PTPRZ1 NM_002851 RAB32 NM_006834 RDBP
NM_002904 RORA NM_002943 RPS24 NM_001026 SCYA15 NM_032964 SCYA17
NM_002987 SCYA3 NM_002983 SCYA4 NM_002984 SCYA5 NM_002985 SCYC1
NM_002995 SDH NM_004168 SELL NM_000655 SERPINB1 NM_030666 SET
NM_003011
[0098]
6 TABLE 6 Unigene Symbol RefSec(GenBank) No. SF3A3 NM_006802 SFRS10
NM_004593 SFRS5 NM_006925 SLA NM_006748 SPP1 NM_000582 SRM300
NM_016333 STAT1 NM_007315 STIP1 NM_006819 TAF2S NM_006706 TANK
NM_004180 TAP1 NM_000593 TBXA2R NM_001060 TIEG NM_005655 TIMP1
NM_003254 TNFRSF1A NM_001065 TNFRSF1B NM_001066 TNFSF10 NM_003810
TNFSF5 NM_000074 UROD NM_000374 USF2 NM_003367 USP12 AF022789 USP7
NM_003470 XBP1 NM_005080 YME1L1 NM_014263 YR-29 NM_014886
[0099] The present invention also provides a method and a system of
analysis using an oligonucleotide array comprising oligonucleotides
(equivalent to (1)) immobilized thereon, each having a nucleotide
sequence comprising at least 20 consecutive nucleotides in genes
that are expressed specifically in Th cells or a complementary
sequence thereof. Such oligonucleotides include those derived from
genes, for example, in terms of Symbol designations of the Unigene,
CD2, CD4, CD28, CD3D, CD3E, CD3G, CD3Z, CD8A, CD62L, CTLA4, MIC2,
SEMA4D, THY1, TCRA, and TCRB. Also, CD44 is known as a gene that is
expressed in Th cells. Examples of these genes are shown in Table 7
together with their accession numbers in the GenBank.
7 TABLE 7 Unigene Symbol RefSec(GenBank) No. CD2 NM_001767 CD28
NM_006139 CD3D NM_000732 CD3E NM_000733 CD3G NM_000073 CD3Z
NM_000734 CD8A NM_001768 CTLA4 NM_005214 MIC2 NM_002414 SEMA4D
NM_006378 THY1 NM_006288 TRA@ M12959 TRB@ X00437
[0100] The present invention also provides a method and a system of
analysis using an oligonucleotide array comprising oligonucleotides
(equivalent to (2)) immobilized thereon, each having a nucleotide
sequence comprising at least 20 consecutive nucleotides in genes
that are not expressed very much in Th cells but are expressed in
cells other than T cells, such as B cells or monocytes, or a
complementary sequence thereof.
[0101] Such oligonucleotides include those derived from genes, for
example, in terms of Symbol designations of the Unigene, ADAM8,
ANPEP, BST1, CD14, CD19, CD22, CD33, CD34, CD36, CD68, CD72, CD74,
CD79A, CD79B, CD81, CD86, CEACAM1, CEACAM3, CEACAM4, CEACAM5,
CEACAM8, CSF1R, CSF2RA, CSF2RB, CSF3R, FCGR1A, FCGR3A, FCGR3B,
FLT3, GP1BA, GP1BB, GP5, GP9, ICAM2, IGSF2, IL3RA, IL5RA, IL12,
IL27, ITGA2, ITGAM, ITGAV, ITGB3, ITGB4, KIT, LAMP1, LAMP2, LRP1,
MS4A2, MST1R, NCAM1, PECAM1, PLAUR, PVR, SELP, SEMA7A, and VCAM1.
Examples of these genes are shown in Tables 8 and 9 together with
their accession numbers in the GenBank.
8 TABLE 8 Unigene Symbol RefSec(GenBank) No. ADAM8 NM_001109 ANPEP
NM_001150 BST1 NM_004334 CD14 NM_000591 CD19 NM_001770 CD22
NM_001771 CD33 NM_001772 CD34 NM_001773 CD36 NM_000072 CD68
NM_001251 CD72 NM_001782 CD74 NM_004355 CD79A NM_001783 CD79B
NM_000626 CD81 NM_004356 CD86 NM_006889 CEACAM1 NM_001712 CEACAM3
NM_001815 CEACAM4 NM_001817 CEACAM5 NM_004363 CEACAM8 NM_001816
CSF1R NM_005211 CSF2RA NM_006140 CSF2RB NM_000395 CSF3R NM_000760
FCGR1A NM_000566 FCGR3A NM_000569 FCGR3B NM_000570 FLT3 NM_004119
GP1BA NM_000173
[0102]
9 TABLE 9 Unigene Symbol RefSec(GenBank) No. GP1BB NM_000407 GP5
NM_004488 GP9 NM_000174 ICAM2 NM_000873 IGSF2 NM_004258 IL3RA
NM_002183 IL5RA NM_000564 ITGA2 NM_002203 ITGAM NM_000632 ITGAV
NM_002210 ITGB3 NM_000212 ITGB4 NM_000213 KIT NM_000222 LAMP1
NM_005561 LAMP2 NM_002294, NM_013995 LRP1 NM_002332 MS4A2 NM_021950
MST1R NM_002447 NCAM1 NM_000615 PECAM1 NM_000442 PLAUR NM_002659
PVR NM_006505 SELP NM_003005 SEMA7A NM_003612 VCAM1 NM_001078
[0103] The present invention also provides to a method and a system
of analysis using an oligonucleotide array comprising
oligonucleotides (equivalent to (3)) immobilized thereon, each
having a nucleotide sequence comprising at least 20 consecutive
nucleotides in genes that are expressed equally in Th cells and in
other cells or a complementary sequence thereof. Such
oligonucleotides include, for example, those derived from internal
control genes already contained in the sample from the subject,
such as leucocytes, or those derived from external control genes
prepared by adding a predetermined amount of gene sequences from
plants, microorganisms, insects, or the like that are absent in
humans to the assay sample at the time of assay. Specific examples
include those derived from genes, for example, in terms of Symbol
designations of the Unigene, AP1B1, AP1G1, AP1S1, AP1S2, AP1M2,
CAMK1, CAMK2A, CAMK2B, CAMK2D, CAMK2G, CD28, CD47, CR1, CREB1,
CTLA4, IFNGR1, IL1R1, IL1R2, IL2RB, IL6ST, ITGA1, ITG26, ITGA3,
ITGA4, ITGA5, ITGA6, LAT, MAPK13, MAPK2K4, MAPK2K7, NFKB1, NFKB2,
RAF1, SLC326, TNFRSF1A, TNFRSF1B, and ZAP70.
[0104] In order to evaluate allergy levels, for example, the Th1
cells:Th2 cells ratio should be analyzed with high accuracy.
Accordingly, it is obvious that the DNA fragment, which is supposed
to be complementarily bound to 1 type of gene only should not be
bound to other types of genes (cross hybridization). This becomes
more complicated as the number of genes to be immobilized on one
array increases. Thus, it is very difficult to completely avoid
cross hybridization between genes on the DNA array for screening,
which has 5,000 to several tens of thousands genes. As a result of
analyses of sequence homology based on the BLAST algorithm, when
the number of nucleotides in a DNA fragment to be used as a probe
is not more than 1,000, it was found preferable to place 1,000 to
1,500 types or fewer DNA fragments on one array. Accordingly, if
the DNA array is used to evaluate and diagnose allergy levels, it
is preferable to prepare an array with a minimal number of only
genes that are associated with the action mechanism of the allergy.
It is not preferable to place genes unassociated with the allergy
because it results in increased production costs for probes and
increased prices for the oligonucleotide arrays. Because the number
of oligonucleotide types to be used as probes for the array can be
kept low, oligonucleotides of one type can be immobilized on
several locations as probes. Thus, reliability can be enhanced by
averaging the signal intensities at several locations.
[0105] More specifically, the use of a DNA array is most suitable
for analyzing allergy levels, wherein the DNA array comprises,
immobilized thereon, a minimal number of oligonucleotide probes
respectively derived from:
[0106] (1) genes that are expressed specifically in Th cells;
[0107] (2) genes that are not expressed very much in Th cells but
are expressed specifically in cells other than T cells, such as B
cells and monocytes;
[0108] (3) genes that are expressed equally in Th cells and in
other cells; and
[0109] (4) genes that are expressed differently in Th1 cells and in
Th2 cells.
[0110] In order to position the oligonucleotides having sequences
derived from the above genes or complementary sequences thereof on
an array as probes, it is necessary to determine which portions in
the gene sequences should be set as probes. In these cases, melting
temperature (Tm) and the absence of cross hybridization should be
taken into consideration. In order to hybridize each DNA fragment
that are immobilized on the DNA array with each sample-derived DNA
fragments with high accuracy (or under highly stringent
conditions), the correlation between hybridization temperature (Th)
and Tm of the immobilized DNA fragment is important. Specifically,
the difference between melting temperature and hybridization
temperature of the immobilized DNA fragment should not exceed
30.degree. C. Since cross hybridization occurs due to high homology
between DNA sequences, homology between sample-derived DNA fragment
to be hybridized with specific immobilized DNA fragment and DNA
fragments among sample-derived DNA fragments that are not
inherently hybridized with the immobilized DNA fragments should be
low enough in order to prevent cross hybridization. It is
preferable that sequences which have mini-hairpin molecular
structures, or portions, which are significantly homologous to a
repeated sequence known as the Alu sequence in human gene, are not
present. In addition to computing the homology among gene sequences
immobilized on one array, homology between the DNA sequences of a
given living species and gene sequences of a target living species
registered in GenBank and the like should be computed. Based on the
comparison between the sequences of the candidate DNA fragments
immobilized on the DNA array and the DNA sequences of a given gene
group which may be contained in an assay sample, it is preferable
not to select significantly homologous DNA sequences as DNA
fragments to be immobilized.
[0111] A DNA fragment to be immobilized as a probe can be
synthesized by PCR using any commercially available cDNA library as
a template. The synthesized product is adjusted to a predetermined
concentration (0.1-1.0 .mu.g/.mu.l) and spotted on a poly-lysine or
amino-silane coated slide glass by using a spotter. Thus, an
oligonucleotide array can be prepared. Alternatively, a probe can
be directly synthesized on a support by any known technique in the
art.
[0112] Allergy levels can be assessed using the oligonucleotide
array in the manner described below. At the outset, peripheral
bloods are sampled from several individual volunteers who had no
allergy symptom, and total RNA or messenger RNA are extracted from
the leukocytes. For example, average messenger RNAs from normal
volunteers are pooled together by mixing messenger RNAs of several
volunteers. This pool of messenger RNAs is hereinafter referred to
as "universal control" in this specification. Subsequently,
peripheral blood is sampled from a subject, and messenger RNA is
extracted from leukocytes. Reverse transcription for messenger RNA
obtained from the subject's peripheral blood is carried out using
oligo dT primer, and labelled cDNA is synthesized using Cy5-dCTP or
the like. Regarding messenger RNA of the universal control,
labelled cDNAs having different labels are synthesized using
Cy3-dCTP or the like. The subject's cDNA (Cy5-labelled) and the
universal control cDNA (Cy3-labelled) are mixed with each other and
subjected to the same oligonucleotide array. Thereafter, they are
allowed to hybridize with each other at a predetermined temperature
for a predetermined length of time. Preferably, hybridization
temperature is 45 to 70.degree. C., and hybridization time is for 6
to 18 hours. After the hybridization, the fluorescence intensities
of Cy5 and Cy3 at the sites where each gene had been spotted are
compared by a fluorescence scanner, thereby determining the
difference in the expression levels therebetween.
[0113] The genes of (1), (2), (3), and (4) can be assessed by
RT-PCR or other methods such as Northern hybridization to determine
the Th1 cells:Th2 cells ratio and the differences in the expression
levels of cytokines therefrom. A method for computing the
expression ratio of Th1/Th2 using (1) to (4) above is described
below.
[0114] Several formulae, which are necessary for describing the
correction method used herein, are first defined.
[0115] In this specification, Th1/Th2 is either:
Th1/Th2=the number of Th1 cells/the number of Th2 cells (formula
1)
[0116] or
Th1/Th2=the expression level for one or more genes expressed in Th1
cells/the expression level for one or more genes expressed in Th2
cells (formula 2).
[0117] The abundance ratio of Th cells to monocytes (hereinafter it
may be referred to as ".alpha.") can be defined by formula 3.
Abundance ratio of Th cells to monocytes (.alpha.)=the number of Th
cells/the number of monocytes=(expression level for one or more
genes that are expressed specifically in Th cells/expression level
for one or more genes that are expressed equally in Th cells and in
monocytes)/(expression level for one or more genes that are
expressed specifically in monocytes/expression level for one or
more genes that are expressed equally in Th cells and in monocytes)
(formula 3)
[0118] Th1/Th2 can be determined as follows:
[0119] Specifically, there are presumed to be m number of probes on
a chip substrate. A different gene corresponds to each probe. A
formula is selected in accordance with a flow chart shown in FIG. 9
depending on whether or not the gene "n" to which a given probe
corresponds (described as a "probe gene" in FIG. 9) is expressed
only in Th cells (Th cell-specific), expressed in cells other than
Th cells, expressed only in Th1 cells (Th1 cell-specific), or
expressed only in Th2 cells (Th2 cell-specific).
[0120] As shown in FIG. 9, for example, the formula to be used
varies depending on whether the gene "n" among the m number of
probes is Th cell-specific or not. When it is also expressed in
cells other than Th cells, formulae 4-3 to 4-5 are used.
[0121] When the gene "n" is Th cell-specific and is expressed
specifically in either Th1 cells or Th2 cells, formula 4-6 is
used.
[0122] When the gene "n" is Th cell-specific and is expressed in
both Th1 and Th2 cells, formulae 4-1, 4-2, and 4-5 are used.
[0123] Specific formulae 4-1 to 4-6 are shown below.
Th1(n)=the expression level of gene "n" in Th1 cells (fluorescence
intensity obtained from gene) (formula 4-1)
Th2(n)=the expression level of gene "n" in Th2 cells (fluorescence
intensity obtained from gene) (formula 4-2)
Th1(n)=the expression level of gene "n" in Th1 cells (fluorescence
intensity obtained from gene).times.(.alpha./(.alpha.+1)) (formula
4-3)
Th2(n)=the expression level of gene "n" in Th2 cells (fluorescence
intensity obtained from gene).times.(.alpha./(.alpha.+1)) (formula
4-4)
[0124] If the total number of genes to be assayed is "k," Th1/Th2
is determined by the following formula. 1 Th1 / Th2 = n = 1 k Th1 (
n ) / n = 1 k Th2 ( n ) .times. ( p , q , r ) ( formula 4 - 5 )
[0125] .beta.(p, q, r . . . ) is a function for normalizing Th1/Th2
to be equal to 1 when Th1:Th2 is 1:1, and it is determined by
experiments. When .beta. is experimentally determined, a group of
previously adjusted samples is prepared to bring the Th1:Th2 to a
known ratio, for example, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8,
or 1:9. Each sample adjusted to the known ratio is used to assay
Th1/Th2. Subsequently, the previously adjusted Th1/Th2 ratio is
plotted on the Y axis and the Th1/Th2 ratio obtained by the assay
is plotted on the X axis. Ideally, each point should be plotted on
the Y=X line, although deviations occur in practice. Thus, these
plots are subjected to curve fitting using the method of least
squares and the like to obtain a calibration curve. When curve
fitting is carried out using a linear function, for example, a
calibration curve for Y=pX+q is presumably obtained. p and q are
parameters obtained by fitting. Specifically, formula 4-5 can be
described as formula 4-5' below. 2 Th1 / Th2 = ( n = 1 k Th1 ( n )
/ n = 1 k Th2 ( n ) ) .times. p + q ( formula 4 - 5 ' )
[0126] When the calibration curve obtained by fitting is a
quadratic function (Y=pX.sup.2+qX+r), a cubic function
(Y=pX.sup.3+qX.sup.2+rX+s), or other nonlinear function, formula
4-5 can be similarly modified and used.
[0127] When the gene "n" is Th cell-specific and is expressed
specifically in Th1 cells or in Th2 cells, the following formula
4-6 is used.
Th1/Th2=median (Rank(X.sub.1), . . . , Rank(X.sub.n1))/median
(Rank(Y.sub.1), . . . , Rank(Y.sub.n1)).times..beta.2 (formula
4-6)
[0128] Rank(X.sub.1) is a ranking of the fluorescence intensity of
a probe corresponding to a gene that is Th1 cell-specific,
Rank(Y.sub.1) is a ranking of the fluorescence intensity of a probe
corresponding to a gene that is Th2 cell-specific, median is a
medium value, and .beta.2 is a normalizing parameter similar as
with .beta. in formula 4-5. Formula 4-6 is hereafter described.
[0129] It is hypothesized that there are n1 number of probes
corresponding to Th1 cell-specific genes and that fluorescence
intensities obtained from these probes are respectively X.sub.1,
X.sub.2, . . . X.sub.n1. Also, there are n2 number of probes
corresponding to the Th2 cell-specific genes and fluorescence
intensities obtained from these probes are respectively Y.sub.1,
Y.sub.2, . . . Y.sub.n2. Ideally, the fluorescence intensity
obtained from the Th2 cell sample by the probe corresponding to the
Th1 cell-specific gene is zero, after background noise is
subtracted. On the contrary, the fluorescence intensity obtained
from the Th1 cell sample by the probe corresponding to the Th2
cell-specific gene is zero. Thus, the fluorescence intensity
obtained by the probe corresponding to the Th1 cell-specific gene
is only derived from the Th1 cell sample, and the fluorescence
intensity obtained by the probe corresponding to the Th2
cell-specific gene is only derived from the Th2 cell sample.
Comparison of the averages (or medians) among X.sub.1, X.sub.2, . .
. X.sub.n1 and those among Y.sub.1, Y.sub.2, . . . Y.sub.n2,
however, cannot accurately determine Th1/Th2. This is because the
Th1 cell-specific gene is different from the Th2 cell-specific
gene, therefore, they cannot be simply compared to each other. If a
sufficiently large number of probes are present on the chip and if
probes are continuously present from those with higher fluorescence
intensity to those with lower fluorescence intensity, when
fluorescence intensities are rearranged in descending or ascending
order for probes with higher or lower fluorescence intensities,
genes with equivalent rankings are expected to be expressed at
substantially the same levels among various genes in the cell
sample. When all the probes on the DNA chip are rearranged in
descending order by fluorescence intensity, the order of X.sub.1,
X.sub.2, . . . X.sub.n1 is designated as Rank(X.sub.1), Rank
(X.sub.2), . . . Rank(X.sub.n1), and the order of Y.sub.1, Y.sub.2,
. . . Y.sub.n1 is designated as Rank(Y.sub.1), Rank(Y.sub.2), . . .
Rank(Y.sub.n1). Since there are a plurality of Th1 specific genes
and a plurality of Th2 specific genes, they are respectively
represented by their medians. For example, the median
(Rank(X.sub.1), . . . Rank(X.sub.n1)):median (Rank(Y.sub.1), . . .
Rank(Y.sub.n1)) ratio can be assigned as Th1/Th2. This median Rank
ratio can be more accurate than simply using fluorescence intensity
particularly when the Th1/Th2 ratio is close to 1. It should be
noted that when the Th1/Th2 ratio is significantly different from
1, an accurate ratio may not always be reflected.
[0130] A statistical technique utilizing Bayesian estimation is
described as follows. One type of RNA is previously divided into
two, one of them is labelled with Cy3 while the other is labelled
with Cy5. An experiment in which they are subjected to competitive
hybridization on the same chip (approximately 5 to 10 chips are
required) is hypothesized. This experimental data is equivalent to
the observed value y=(y1, y2, . . . , yi, . . . , yn) obtained by
extracting random samples of size n [Y1, Y2, . . . , Yi, . . . ,
Yn] from a normal population N(c, .sigma..sup.2) in which c=the
average of Cy5/Cy3 is 1.0 while the variance .sigma..sup.2 is
unknown. Since error factors such as differences between chips,
differences between coloring agents, and individual differences in
handling are not correlated between experiments, [Y1, Y2, . . . ,
Yi, . . . , Yn] can be said to be mutually independently and
identically distributed (i.i.d.).
Yi.about.i.i.d.N(c, .sigma..sup.2)=N(1, .sigma..sup.2) (formula
5)
[0131] The ultimate goal of this Bayesian estimation is to estimate
the .sigma..sup.2 for Yi. If .sigma..sup.2 for the population N(1,
.sigma..sup.2) is estimated by this estimation, .sigma..sup.2 for
Yi is identical according to formula 5. In the Bayesian estimation,
.sigma..sup.2 is presumed to have a distribution. Thus, what is
obtained is an estimate for .sigma..sup.2. An average, a mode (a
point of maximal frequency), or a Highest Density Region (HDR) as
an confidence interval can be obtained as the estimate. The 90%
highest density region is so to speak the shortest in the 90%
interval of unknown parameter .sigma..sup.2, and it always contains
a peak value of posterior distribution (posterior mode). In
addition, posterior densities at both ends of the interval are
equal to each other.
[0132] When formula 5 is realized, the joint probabilistic
distribution of [Y1, Y2, . . . , Yi, . . . , Yn] (the probabilistic
distribution which simultaneously fulfills a1<Y1.ltoreq.b1,
a2<Y2.ltoreq.b2, . . . , an<Yn.ltoreq.bn) p(y'.vertline.c,
.sigma.2) can be represented in a form that is multiplied by a
gaussian distribution: 3 p ( y ' | c , 2 ) = i = 1 n ( 1 / 2 ) exp
( - 1 / 2 2 .times. ( yi - c ) 2 ) ( formula 6 )
[0133] wherein .PI. is a multiplication sign. Accordingly, when a
vector of observed value y=(y1, y2, . . . , yi, . . . , yn) is
provided, the likelihood function 1 (.sigma..sup.2.vertline.y) is
either 4 1 ( 2 | y ) i = 1 n ( 1 / ) exp ( - 1 / 2 2 .times. ( y -
c ) 2 ) ( formula 7 )
[0134] or
1(.sigma..sup.2.vertline.y).varies.(.sigma..sup.2)-n/2.times.exp(-ns.sup.2-
/2.sigma..sup.2) (formula 8).
[0135] It should be noted that s.sup.2 is the distribution of
observed values having the population mean c as a center and
represented by the following formula. 5 s 2 = ( 1 / n ) .times. i =
1 n ( yi - c ) 2 ( formula 9 )
[0136] Noninformative prior distribution is hypothesized as the
prior distribution p(.sigma..sup.2) of a variance. This hypothesis
is reasonable in that the arbitrariness for unknown parameters of
prior distribution is eliminated as much as possible, and posterior
distribution is controlled by data as much as possible. In general,
locally uniform prior distribution is employed as noninformative
prior distribution. In locally uniform distributions, unknown
parameters are uniformly distributed at least locally either in the
form of squared, cubed, or logarithmic values in order to represent
the vagueness of prior information. Specifically, it may be set so
as to be in proportion with the square root of the Fisher
information. If the locally uniform distribution is employed as
prior distribution, it may be set as
p(.sigma..sup.2).varies..sigma..sup.-2 (formula 10).
[0137] That is, the prior distribution p(.sigma..sup.2) of
.sigma..sup.2 is .sigma..sup.-2, i.e., a constant. Posterior
distribution is considered as follows. Based on the Bayesian
theory,
p(.sigma..sup.2.vertline.y).varies.1(.sigma..sup.2.vertline.y)p(.sigma..su-
p.2) (formula 11)
[0138] is effected. Thus, the posterior distribution
p(.sigma..sup.2.vertline.y) of .sigma..sup.2 is equal to
.chi..sup.-2(n, ns.sup.2) based on
p(.sigma..sup.2.vertline.y).varies.(.sigma..sup.2)-(n/2+1).times.exp(-ns.s-
up.2/2.sigma..sup.2) (formula 12).
[0139] .chi..sup.2(.nu., .lambda.) is referred to as the inverse
Chi square distribution with a scale parameter of .lambda. and with
.nu. degree of freedom. Since it is known that the average of
.chi..sup.-2(.nu., .lambda.) is .lambda.(.nu.-2) and the mode (a
point of maximal frequency) is .lambda./(.nu.+2), the following
formulae can be considered as point estimates of .sigma..sup.2:
[0140] in the case of employing the average as the standard:
.sigma..sup.2=ns.sup.2/(n-2) (formula 13)
[0141] in the case of employing the mode as the standard:
.sigma..sup.2=ns.sup.2/(n+2) (formula 14).
[0142] Based on formula 12, the correlation
ns.sup.2/.sigma..sup.2.about..chi..sup.2(n) (formula 15)
[0143] can be posteriorly obtained. .chi..sup.2(n) of formula 15 is
the Chi square distribution with n degrees of freedom. ns.sup.2 is
a fixed value (observed value), and .sigma..sup.2 is a random
variable. HDR can be obtained by using formula 15 and a
mathematical table. When the Th1/Th2 ratio is obtained by using
formulae 13, 14, and 15, the degree of statistically significant
differences can be assessed by comparing these obtained values
against 1.0 (Th1/Th2 value of a control).
[0144] For example, if y.sub.1=1.4, y.sub.2=0.89, y.sub.3=1.24,
y.sub.4=0.91, and y.sub.5=1.04 were obtained in 5 experiments,
s.sup.2=0.04788. According to point estimation (formula 13) on the
basis of the averages, Yi.about.N(1, 0.0798) is obtained. According
to point estimation (formula 14) on the basis of the modes,
Yi.about.N(1, 0.0342) is obtained. According to formula 15,
.sigma..sup.2.about.0.2394.chi..sup- .-2(5) is obtained. Thus, 90%
HDR of .sigma..sup.2 is found to be 0.019 to 0.177 by using a
mathematical table.
[0145] Bayesian estimation represented by formula 5 to formula 15
is one statistical technique. Accordingly, methods other than
Bayesian estimation, for example, Neyman-Pearson's estimation may
be employed.
[0146] With the use of this Bayesian estimation, for example, when
the Th cells ratio in all leukocytes is previously fixed at X (1.0
in the example above) and then the observed Th cells ratio is Y,
the degree of statistically significant differences in the Th cells
ratio can be computed. The ratios of T cells, B cells, and the like
in all leucocytes are also valuable in terms of assessing the
subject's physical condition. If there is any significant change in
the Th cells ratios in all leucocytes, the subject can be diagnosed
as previously having had some changes in his/her physical
condition.
EXAMPLES
[0147] One example of the present invention, which simulates an
example for assessing changes in the subject's allergy level, is
described. Since it is difficult to obtain blood from an allergy
patient, Th1 cells, Th2 cells, and monocytes are separated and
extracted from peripheral blood of a normal volunteer, and the
mixing ratios of these cells are varied to simulate the examination
using whole blood. Monocytes express many genes similar to Th
cells, and thus, there present a major bias in the assay using
whole blood. If the Th1 cells:Th2 cells ratio can be accurately
determined even when monocytes are present, the bias derived from
the existence of B cells and the like can be similarly eliminated
and the assay can be accurately carried out even when whole blood
is assayed.
[0148] The observed Th1/Th2 values are described, (1) when a sample
containing only Th1 and Th2 cells is assayed and (2) when a sample
containing monocytes besides Th1 and Th2 cells is assayed. The
mixing ratios of Th1 cells and Th2 cells were previously set at
1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1.
[0149] Th1/Th2 is represented by formula 1 or formula 2. In the
examples herein, the definition of formula 1 is used when preparing
samples. The definition of formula 2 is used for observed values.
In general, formula 1 and formula 2 are in agreement when the gene
expression level per cell does not vary between each subject of the
examination. Formula 1 and formula 2, however, are not always in
agreement when, for example, Th cells previously had genetic
abnormalities and the expression level of a certain cytokine
differs from a normal level. In this specification, formula 1 and
formula 2 were substantially in agreement as a result of the use of
the blood of a normal volunteer, however, the results on a normal
volunteer as such cannot be always applicable for some specific
diseases. In these cases, the present invention may be applied for
each disease after formula 1 and formula 2 are independently
evaluated.
[0150] The expression levels of the genes that are expressed
specifically in monocytes, correspond to those of the genes that
are not substantially expressed in Th cells but are expressed
specifically in cells other than T cells, such as B cells or
monocytes. For example, the abundance ratio of Th cells to B cells
can be similarly determined as formula 3.
[0151] In the description given hereinafter, as the expression
levels of genes that are expressed specifically in Th cells, the
average fluorescence intensities obtained from a plurality of genes
that are expressed specifically in Th cells is employed. Similarly,
the average fluorescence intensities obtained from a plurality of
genes that are expressed specifically in monocytes is employed as
the expression level for genes that are expressed specifically in
monocytes. The average fluorescence intensities obtained from a
plurality of genes that are expressed equally in both Th cells and
monocytes is employed as the expression level for genes that are
expressed equally in both Th cells and monocytes.
[0152] When assaying Th1/Th2 based on the assay result using DNA
chips, a method can be utilized in which only the genes that are
expressed differently in Th1 cells and in Th2 cells, and are
expressed specifically in Th cells are used. Genes that are
expressed specifically in Th cells are, for example, CD2, CD28,
CD3D, CD3E, CD3G, CD3Z, CD8A, CTLA4, MIC2, SEMA4D, THY1, TRA@, and
TRB@ (Symbol designations of the Unigene). However, since only a
small number of genes fall under this range, there were relatively
large variations in the observed values as a result of our data
analysis. In this example, therefore, Th1/Th2 was determined by
formula 4.
[0153] As annotation of the human genome progresses, in the future,
it can be expected that the number of genes, which belong to genes
that are expressed differently in Th1 cells and in Th2 cells and
are expressed specifically in Th cells, would increase to the
extent that assay variation can be sufficiently lowered, for
example 50 to 200. In that case, the use of genes that are
expressed differently in Th1 cells and in Th2 cells and are
expressed specifically in Th cells may result in achieving
sufficient assay sensitivity and reproducibility. In such cases,
only formula 4-1 and formula 4-2 may be used. The experimentation
methods are described as follows:
[0154] Formulae 1 to 4 in this specification were computed by using
all the genes as shown in Tables 1 to 9.
[0155] 1. Acquisition of Th1 Cells and Th2 Cells from Blood
[0156] Whole blood (50 ml) was sampled from a normal volunteer with
a heparin-coated hypodermic needle. Serum free RPMI 1640 medium
(supplemented with 1% penicillin/streptomycin) (50 ml) was added to
the whole blood and diluted. This diluent was poured into a 50 ml
centrifuge tube containing 10 ml of Ficoll-Paque, and
centrifugation was carried out at room temperature at 800 g for 30
minutes. Peripheral blood mononuclear cells (PBMC) were carefully
sampled from the interface and washed twice with Phosphate Buffered
Saline (PBS). Monocytes were removed by depositing onto a gelatine
coated tissue culture flask. RPMI 1640 medium+10% FCS+5 .mu.g/ml
PHA was added to PBMC. IL 12 (1 ng/ml) and anti-IL4 antibody (0.1
.mu.g by diluting 500 .mu.g to 5,000-fold) were added to the PBMC
medium mentioned above, and Th1 cells were cultured therein. Medium
was changed every 3 days. IL4 (50 ng/ml) and anti-IL12 antibody
(0.1 .mu.g by diluting 500 .mu.g to 5,000-fold) were added to the
PBMC medium mentioned above, and Th2 cells were cultured therein.
Medium was changed every 3 days. Under these culture conditions,
approximately two thirds of the cells were CD4+ cells and the
remaining one third was CD8+ cells. Monocytes, B cells, and NK
cells were not substantially present. In order to remove CD8+
cells, anti-CD8+ monoclonal antibodies were added, and incubation
was carried out at 4.degree. C. for 30 minutes. Thereafter, unbound
monoclonal antibodies were removed and the remaining mixture was
bound to magnetic beads whose surfaces had been coated with a goat
anti-mouse Fc-specific antibody. The ratio of the number of
beads:the number of cells was 25:1. CD8+ cells were removed by a
bead trap using magnetic force, and the residual CD8+ ratio was not
more than 1%. The thus obtained Th1 cells and Th2 cells were
immediately used or cryopreserved in liquid nitrogen. The
composition of the preserved solution was 95% medium/5% DMSO.
[0157] 2. Acquisition of Monocytes from Blood
[0158] Whole blood (50 ml) was sampled from a normal volunteer with
a heparin-coated hypodermnic needle. Serum free RPMI 1640 medium
(supplemented with 1% penicillin/streptomycin) (50 ml) was added to
the whole blood and diluted. This diluent was poured into a 50 ml
centrifuge tube containing 10 ml of Ficoll-Paque, and
centrifugation was carried out at room temperature at 800 g for 30
minutes. PBMC was carefully sampled from the interface and washed
twice with PBS. Thereafter, RPMI 1640 medium+7.5% FCS+100 .mu.g/ml
streptomycin and 100 U/ml penicillin were added to the PBMC
culture. PBMC was mixed with magnetic beads whose surfaces had been
coated with anti-CD14 monoclonal antibody, and the resulting
mixture was incubated and separated by a bead trap using magnetic
force. The separated monocytes were cultured at 37.degree. C. in a
5% CO.sub.2 incubator for 30 minutes, and high purity monocytes
were obtained. Positive staining using CD14 and flow cytometry were
carried out. As a result, 99% or more were confirmed to be
monocytes. The thus obtained monocytes were immediately used or
cryopreserved in liquid nitrogen. The composition of the preserved
solution was 90% medium/10% DMSO.
[0159] 3. Acquisition of Total RNA from Th Cells and Monocytes and
Preparation of RNA Mixture
[0160] Total RNA was obtained from the cells using ISOGEN (Nippon
Gene Co., Ltd.). Specifically, 750 ml of ISOGEN was added to
5.times.10.sup.6 Th1 cells, Th2 cells, or monocytes, respectively.
This process is the process in which a cell membrane is disrupted
to collect nucleic acid from the cell and phenol extraction is
performed. After centrifugation was carried out at 4.degree. C. at
15,000 rpm for 30 minutes, the supernatant was carefully collected
with a pipette and transferred to a new Eppendorf tube. Thereafter,
the following were carried out: chloroform extraction twice,
isopropyl precipitation once, and rinsing with 70% ethanol once.
The RNA, which remained at the bottom of the Eppendorf tube, was
dissolved in TE buffer. The RNA concentration was determined by
measuring absorption. OD260/OD280 was 1.9 to 2.0. The thus obtained
Th1 cell-derived total RNA and Th2 cell-derived total RNA were
mixed with each other at 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2,
and 9:1 based on the weight ratio of RNA. In the case where
monocytes were added, the abundance ratio of T cells to monocytes
in leukocytes were taken into consideration to bring the weight
ratio of RNA derived from both cells to Th1 cells or Th2
cells:monocytes=5:4. This ratio is equivalent to the ratio of
5.times.10.sup.8, which is half the number of T cells,
1.times.10.sup.9, in 1 liter of human blood, and 4.times.10.sup.8,
which is the number of monocytes and/or macrophages in 1 liter of
human blood.
[0161] 4. Preparation of DNA Array
[0162] Subsequently, 793 oligonucleotide probes, which have high
specificity and identical Tm, were designed in accordance with the
algorithm comprising the steps of:
[0163] 1. reading a gene sequence file;
[0164] 2. inputting experimental conditions on, for example, salt
concentration and hybridization;
[0165] 3. inputting a length range of DNA fragments to be
immobilized;
[0166] 4. calculating the melting temperature of each candidate DNA
fragment for immobilization and eliminating from the candidate list
the DNA fragments whose melting temperatures are outside a given
range;
[0167] 5. eliminating from the candidate list DNA fragments, which
have a short sequence having specific higher order structure or
short repeated sequence;
[0168] 6. eliminating from the candidate list DNA fragments, which
are highly homologous to repeated sequences such as the Alu
sequence;
[0169] 7. eliminating from the candidate list DNA fragments, which
are highly homologous to other gene sequences;
[0170] and the like. In addition to these 793 types of human gene
probes, 3 types of oligonucleotide sequences (lambda DNA, pUC18
plasmid DNA, and M13mp18 DNA), which are absent in humans, were
added as external control genes for proofreading to prepare 796
types of oligonucleotides, and these were immobilized on a glass
substrate in the manner as disclosed below. When immobilizing on
the glass substrate, DNA fragments were positioned, for example, as
shown in FIG. 1. In FIG. 1, the following are separately
immobilized: probe DNA of genes that are expressed specifically in
Th cells (12), probe DNA of genes that are not expressed very much
in Th cells but are expressed specifically in cells other than T
cells, such as B cells and monocytes (13), probe DNA of genes that
are expressed equally in Th cells and in other cells (14), and DNA
fragments (probe DNA) having gene sequences that are expressed
differently in Th1 cells and in Th2 cells or complementary sequence
thereof (11). Further, the following are separately immobilized:
probe DNA (111) of genes that are expressed differently in Th1
cells and in Th2 cells and whose expression level in Th1 cells is
higher than that in Th2 cells, and probe DNA (112) of genes that
are expressed differently in Th1 cells and in Th2 cells and whose
expression level in Th2 cells is higher than that in Th1 cells.
[0171] Prior to the immobilization of the probes, a commercially
available slide glass (Gold Seal Brand) was first immersed in an
alkaline solution for 2 hours at room temperature. Thereafter, the
slide glass was transferred into distilled water and rinsed 3 times
to completely remove the alkaline solution. Subsequently, the
rinsed slide glass was immersed in an aqueous solution of 10%
poly-L-lysine (Sigma) for 1 hour, the slide glass was then pulled
out, and the aqueous solution of poly-L-lysine was removed by
centrifugation for 1 minute by using a centrifuge for micro titer
plates. Thereafter, the slide glass was put into a vacuum incubator
and dried at 40.degree. C. for 5 minutes, to introduce an amino
group on the slide glass.
[0172] An automatic DNA synthesizer was used to synthesize
oligonucleotides, and the oligonucleotides were then purified by
High-Performance Liquid Chromatography. Subsequently, the 1 .mu.l
of synthesized and purified oligonucleotide (concentration of 2
.mu.M), additives, and the like were mixed to prepare a spotting
solution. The prepared spotting solution was spotted on the slide
glass at desired positions using a spotter. FIG. 2 shows a general
structure of a DNA chip. As shown in FIG. 2, fluorescence-labelled
gene 23 hybridizes with DNA probe 22 immobilized on support 24 in a
sequence-specific manner, and the fluorescence emitted from the
bound label is detected by fluorescence detector 21.
[0173] 5. Assay Result
[0174] FIG. 3 shows an example in which a sample containing only
Th1 and Th2 cells is assayed to evaluate Th1/Th2, and FIG. 4 shows
an example in which a sample containing monocytes in addition to
Th1 and Th2 cells is assayed to evaluate Th1/Th2. FIG. 3(A) is a
result obtained when Th1/Th2 was evaluated by using only genes that
are expressed differently in Th1 cells and in Th2 cells. FIG. 3(B)
is a result obtained, using formula 4 (formulae 4-1 to 4-6), when
the following genes were used: genes that are expressed differently
in Th1 cells and in Th2 cells, genes that are expressed
specifically in Th cells, genes that are not expressed very much in
Th cells but are expressed specifically in cells other than T
cells, such as B cells and monocytes, and genes that are expressed
equally in Th cells and in other cells. FIG. 4(A) and FIG. 4(B) are
the similar as FIG. 3. In the graphs of FIG. 3 and FIG. 4, the X
axis indicates the observed values and the Y axis indicates the
expected values. The expected values represent Th1/Th2 (formula 1)
determined from the mixing ratio of Th1 cells and Th2 cells, and
the observed values represent Th1/Th2 (formula 2) determined from
the gene expression level. As shown in both (A) and (B) in FIG. 3,
Th1/Th2 was substantially equally assayed from the sample
containing only Th1 and Th2 cells. Tis is because
.alpha./(.alpha.+1) in formula 4 is almost 1. As shown in FIG.
4(A), however, if the sample contains cells other than Th cells, in
this case, monocytes, Th1/Th2 cannot be accurately assayed and
assay variation is also large. This is because .alpha./(.alpha.+1)
is not equal to 1. In this example, Th1 cells or Th2
cells:monocytes=5:4, i.e, .alpha.=5/4, and thus,
.alpha./(.alpha.+1)=0.555. The assignment of this value 0.555 into
formula 4-5 and the use of the normalizing parameter .beta.=0.92
separately obtained when Th1:Th2 is 1:1 enables the substantially
accurate assay of Th1/Th2 as shown in FIG. 4(B). This can be
realized because contribution of monocytes-derived gene expression
is corrected by formula 4. Thus, Th1/Th2 can be assayed without the
isolation of Th cells. This process enables the experiments to be
carried out in a simpler manner, at lower cost, and more
rapidly.
[0175] In the present invention, changes in the expression levels
of: (i) genes that are expressed differently in Th1 cells and in
Th2 cells; as well as (ii) genes that are expressed specifically in
Th cells; (iii) genes that are not expressed in Th cells; and (iv)
genes that are expressed equally in Th cells and in other cells,
were assessed, and in particular, the value of (i) was corrected by
using values of (ii), (iii), and (iv). This improved the assay
accuracy, and has led to the completion of the present
invention.
[0176] Th1 cells and Th2 cells are associated with various immune
disorders. For example, Th1 cells are known to significantly
infiltrate into synovial fluids of patients afflicted with
rheumatoid arthritis, and Th2 cells are known to selectively
infiltrate into skins of patients afflicted with atopic dermatitis.
Compared to a normal volunteer, observed in the blood of patients
with atopic dermatitis are an increase in the number of Th2 cells
which express CCR4 and a decrease in the number of Th1 cells which
express CXCR3, (Murai and Matsushima, Saibou Kougaku (Cell
Technology), Vol. 19, 703-707, 2000). In addition, strong
association between Th1/Th2 imbalance and asthma, AIDS,
tuberculosis, Lyme disease, type I diabetes, rheumatoid arthritis,
multiple sclerosis, and the like have been pointed out. It is
expected that controlling the Th1/Th2 balance can prevent and treat
these diseases.
[0177] There are several cytokines generated in Th1 cells, for
example, IFN-gamma and IL2. Also, there are several cytokines
generated in Th2 cells, for example, IL4, IL5, IL6, IL9, IL10, and
IL13. Among these several cytokines, the IFN-gamma: IL4 ratio is
said to be the closest to Th1 cells:Th2 cells ratio. There are,
however, quite a number of immune disorders associated with normal
IFN-gamma:IL4 ratios. For example, in idiopathic nephrotic
syndrome, which is presumed to be deeply associated with atopic
dermatitis, there is no difference in the IFN-gamma mRNA:IL4 mRNA
ratio in the peripheral blood cell between patients and normal
volunteers. However, the level of IL13 mRNA in the peripheral blood
mononuclear cell is significantly increased in patients (Yap, H-K
et al., Th1 and Th2 cytokine mRNA profiles in childhood nephrotic
syndrome: Evidence for increased IL-14 mRNA expression in relapse,
J. Am. Soc. Nephrol 10, 529-537, 1999). This indicates that even
though there is no difference between the number of Th1 cells and
the number of Th2 cells, the level of produced cytokines is
imbalanced if either Th1 cells or Th2 cells have abnormalities.
Since the DNA microarray enables the observation of a large number
of genes associated with the Th1/Th2 balance at the same time, it
can deal with various immune disorders and a cost required for one
diagnosis can be lowered.
EFFECT OF THE INVENTION
[0178] The present invention provides a method and a system for
allergy analysis by using RNA derived from leukocytes extracted
from peripheral blood as such, correcting, assessing the balance in
helper T (Th) cells (Th1 cells:Th2 cells ratio-(Th1/Th2)), and
comparing the balance with the number of Th1 cells/the number of
Th2 cells in a nucleic acid sample or the Th1/Th2 data of a patient
and of a normal volunteer. The present invention can also provide a
method and a system for allergy analysis using a highly
reproducible and reliable array having a minimal number of DNA
fragments (oligonucleotides) thereon, which is realized by
identifying essential gene groups for the Th1/Th2 assay.
[0179] All publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
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